Not applicable.
Affinity chromatography is a powerful method used to purify molecules and pharmaceutical compounds. A wide variety of molecules have been purified using various affinity purification schemes. Generally, the synthesis of affinity chromatography supports involves coupling a ligand having an affinity for the molecule being purified to a chromatography support. Through the specific interactions between the molecule and its ligand, significant increases in the purity of the molecule over the starting material can be achieved or significant amounts of a molecule can be removed from a starting material.
Typically, the synthesis of affinity chromatography resins involves reacting a ligand with a functional group on a chromatographic support or a derivatized group on a chromatographic support to form a ligand-matrix bond. Unfortunately, not all ligand-chromatographic support bonds are stable and the chromatographic support itself may trap ligands (or complexes of ligands) that are not bound to a functional group. Furthermore, these ligands can leach from the column during purification, contaminating the product. This is often a serious concern, especially in the pharmaceutical arena. Often regulatory agencies will issue strict guidelines to pharmaceutical companies for the amount of ligand that can be present in a pharmaceutical composition; during the regulatory approval process and on approved drugs, regulatory agencies (e.g., the FDA in the US) require that stringent limits (or specifications) be met on the amounts of contaminants such as ligands that can be present in the pharmaceutical. Thus, the presence of these contaminants can result in a ruined batch of the pharmaceutical or in increased expenditures of time and effort to further purify the pharmaceutical away from the ligand. Moreover, immunization of patients against the drug or anaphylactic shock could result if the ligand contaminant is an antibody and the molecule is used in a clinical setting (Ubrich and Rivat (1996) Art. Cells, Blood Subs., and Immob. Biotech. 24: 65-75). Therefore a need in the art exists for methods of decreasing the amount of ligand that leaches from an affinity column during purification of the molecule of choice. The present invention fulfills these and other needs.
The present invention provides methods for conditioning a chromatography resin after storage. The methods involve contacting a chromatography resin with an effective amount of a hydroxyalkyl amine compound that comprises at least one hydroxyl moiety. An example of a suitable compound is ethanolamine. The methods are generally used on a chromatography resin that is composed of a ligand that was conjugated to a chromatographic support at least 48 hours prior to contacting the chromatography resin with the hydroxyalkyl amine compound. For example, the invention provides methods for conditioning a chromatography resin that consists of an activated hydroxyl chromatographic support to which is attached an affinity ligand.
The invention also encompasses methods for preparing an affinity resin from which less ligand will leach during purification of a molecule to which the ligand binds. The methods entail contacting an affinity resin with a hydroxyalkyl amine compound. The affinity resin is composed of a ligand that is conjugated to a chromatographic support, and has been stored for at least 48 hours after coupling the ligand to the chromatographic support. The affinity ligand is incubated in the presence of the hydroxyalkyl amine compound for a time sufficient to remove ligand that is not stably bound to the chromatography resin. Sufficient unstably bound ligand is removed by the treatment so that when the treated affinity resin is used for affinity purification of a molecule to which the ligand specifically binds, less ligand leaches from the affinity resin as compared to a purification of the molecule on the affinity resin that has not been treated with the compound.